ABSTRACT The lack of effective therapy for metastatic triple negative breast cancer (TNBC) prompted us to develop an effective strategy for this malignancy. In our strategy T cells genetically engineered with tumor antigen-specific chimeric antigen receptor (CAR) are the effectors.. In experiments which are under way we are using the membrane bound chondroitin sulphate proteoglycan (CSPG)4 as a target, since this tumor antigen is expressed in about 60% of TNBC tumors and has a restricted distribution in normal tissues. The experiments in progress suggest that CSPG4-specific CAR T cell-based immunotherapy is effective in eradicating human TNBC cells grafted in mouse models. These results have prompted us to test whether human TNBC cells express other tumor antigens which can be targeted with CAR. This would allow us to apply CAR T cell-based immunotherapy in TNBC patients who do not express CSPG4 in their tumors. We have found that B7-H3 is expressed in about 70% of TNBC tumors independently of CSPG4. Furthermore B7-H3 can mediate the recognition of TNBC by CAR T cells. B7-H3 has several additional attractive features for its use as a target of CAR T cell-based immunotherapy, since i) it is expressed both by differentiated TNBC cells and by TNBC cancer initiating cells; ii) it is expressed both in primary and metastatic TNBC tumors and it has a restricted distribution in normal tissues. However B7- H3 is expressed on dendritic cells. Dendritic cells are recognized by T cells transduced with the CAR generated from our B7-H3-specific mAb 376.96. This reactivity raises the question whether the targeting of dendritic cells by B7-H3-specific CAR T cells is incompatible with their clinical application for the treatment of malignant diseases because of the negative impact on the host?s ability to mount an immune response. To address this question we will take advantage of the cross-reactivity with mouse B7-H3 of the human B7-H3-specific mAb 376.96, from which the CAR used in the proposed studies has been generated. This allows us to test safety of B7-H3 CAR-T cells in an immunocompetent mouse model. Therefore the specific aims of this grant application will test the hypotheses that i) B7-H3-specific CAR mouse T cells do not have major detrimental effects when injected to syngeneic immunocompetent mice, and ii) B7-H3 CAR human T cells are able to eradicate metastases in NSG mice orthotopically grafted with human TNBC cells and then subjected to surgical removal of the primary tumors. The results generated by the outlined studies will contribute to determine the safety and anti-tumor activity of B7-H3 specific CAR T cells for the treatment of TNBC.